In the present study, we examined the effect of a PAR1 agonist on DSS-treated colons. DSS-treatment caused significant shortening of colons and decreased body weight. The transient hyperpolarization response to thrombin observed in control colonic muscles was greatly decreased in DSS-treated colons. Similarly, the initial relaxations observed in response to thrombin in control colonic muscles were changed by DSS-treatment to an increase in contractions without a relaxation phase. We reported previously that the initial hyperpolarization and relaxation responses to thrombin are due to activation of SK3 channels in PDGFRα+ cells14. In the present study we found that depolarization and contractile responses to apamin were decreased significantly after DSS-treatment in comparison to untreated colons. The depolarization phase of responses to thrombin is due to activation of ANO1 conductance in ICC14. Thus, we examined the effects of thrombin on membrane potentials in the presence of apamin. Thrombin induced less depolarizations in DSS-treated muscles than in control muscles in the presence of apamin. These differences were consistent with changes we observed in expression of key genes in PDGFRα+ cells and ICC. Pdgfra and Kcnn3 were downregulated in cells from DSS-treated colons, but no statistically significant change was observed in Kit and Ano1. Seemingly at odds with the electrophysiological observations, contractile responses to thrombin increased significantly in colonic muscles after DSS-treatment and in the presence of apamin. This uncoupling between electrical and mechanical responses appeared to be due to enhanced expression of the main proteins responsible for Ca2+ sensitization responses in DSS colitis.
IBD results from multifactorial causes including genetic, immune, environmental and endogenous factors, such as defects in barrier function, vascular supply, or enteric nerve function17. Proteinases that activate PAR are found in increased concentrations in IBD patients18,19. Among a variety of chemically-induced animal models of colitis, DSS-induced colitis model has been used because it is simple and similar to human ulcerative colitis20. In the present study, we used DSS-induced colitis to examine how responses to a PAR1 agonist are remodeled in colitis.
PAR1 is a G protein–coupled receptor activated by thrombin. PAR1 activation mediates several effects of thrombin other than platelet aggregation, including inflammation. PAR1 activation induces changes in vascular tone, increased vascular permeability, and granulocyte chemotaxis21,22. In the gut, PAR1 is expressed by a variety of cell types, including enterocytes; endothelial cells; enteric neurons, SMCs, ICC and PDGFRα+ cells14,15,23. Intestinal motility is modulated by PAR1 activation. PAR1-activating peptides reduce spontaneous contractions or causes a biphasic response: initial relaxation followed by contraction in rat intestine 24,25. In the mouse gastric fundus, responses to PAR1 activation are also biphasic characterized by relaxation that masks concomitant contractile effects26. PAR1 activation also causes biphasic contractile effects in the colon14,15,23−25. A study in vivo showed that administration of a PAR1 agonist increased gastrointestinal transit 27, thus demonstrating that PAR1 activation can affect generalized motility patterns. The integrated effects of PAR1 activation on GI motility might depend on the cellular target that the PAR1 agonist first reaches. PDGFRα+ cells and ICC are both responsive to PAR agonists14. SMCs may also contribute to PAR responses via Ca2+-sensitization mechanisms intrinsic to these cells15. Electrophysiological pacemaker activity, post-junctional neural regulation and excitation-contraction coupling in SMCs that powers GI motility result from the combined actions of at least 3 types of cells, including SMCs, ICCs and PDGFRα+ cells. We have referred to these electrically coupled cells as the SIP syncytium3. For example, activation of SK3 channels, which are highly expressed in PDGFRα+ cells, causes hyperpolarization of cells in the SIP syncytium and decreases the excitability of SMCs9,10. In contrast, activation of ANO1 channels, which are expressed dominantly in ICC, depolarizes the cells of the SIP syncytium and increases the excitability in SMCs28,29. Thus, changes in expression of key conductances in SIP cells can influence excitability and possibly upset normal GI motility patterns. In this study, we dissected how DSS treatment affects cellular mechanisms that mediate responses to PAR1 and regulate contractile behaviors of colonic SMCs.
RMPs were depolarized in DSS-treated colonic muscles in comparison colonic muscles from control mice. We also found that thrombin-induced hyperpolarization, which is inhibited by apamin and therefore mediated by activation of SK channels14, was also reduced in DSS-treated colons. Molecular data showed the downregulation of Pdgfra and Kcnn3 which supported the functional data. Depolarization and the excitatory phase of PAR responses are mediated by ICC and due to activation of ANO1 channels, as this conductance is expressed in ICC and blocked by 5-nitro-2-(3-phenylpropyl-amino) benzoic acid (NPPB)29,30. Thrombin induces a transient relaxation in control colonic muscles. The relaxation phase of the thrombin response was absent in muscles from DSS-treated colon, and this was associated with downregulation of SK channels in PDGFRα+ cells. If the balance between depolarizing (activation of ANO1 channels in ICC) and hyperpolarizing (activation of SK3 channels in PDGFRα+ cells) is disrupted, as occurs in DSS colitis, one might expect greater depolarization and greater contractile responses to PAR1 activation. Greater contractile responses were observed in our experiments, but we did not observe statistically greater depolarization responses in muscles DSS-treated colons, even when residual SK3 channels were blocked by apamin. This suggests that other factors may be involved in shaping electrophysiological responses to thrombin. The increase in contractions in response to thrombin in DSS-treated colonic muscles also seemed to be due to additional factors, such as upregulation of Ca2+ sensitization mechanisms in SMCs.
PAR activation by thrombin also increases MYPT1 phosphorylation by ROCK, as previously reported15. Thrombin treatment significantly increased MYPT1 T853 phosphorylation in simian colonic muscles. CPI-17 T38 phosphorylation which is typically associated with Ca2+ influx. Apamin pretreatment inhibited hyperpolarization and increased CPI-17 T38 phosphorylation. Since CPI-17 can be phosphorylated by PKC5,31,32, Gö 6976, the inhibitor of Ca2+-dependent PKCs, blocked CPI-17 T38 phosphorylation in response to thrombin15. These previous reports suggest that thrombin also induces Ca2+ influx or release in SMCs and activates PKC to induce CPI-17 T38 phosphorylation. In the present study, we found that that expression of MYPT1 and CPI-17 proteins were increased in DSS-induced colitis, possibly increasing the Ca2+ sensitization mechanisms in SMCs. This remodeling may have contributed to the apparent uncoupling between membrane potential responses and contractions.
In conclusion, these studies showed that responses to the PAR1 agonist, thrombin, are remodeled in DSS colitis. Relaxation responses are abolished due to decreased expression of SK3 in PDGFRα+ cells and contractions are increased due to loss of the hyperpolarizing phase of the response to PAR1 activation and upregulation of proteins involved in Ca2+ sensitization. These cell specific alterations may provide a partial explanation for some of the dysmotility observed in colitis.